The inhibition of dopamine reuptake via the dopamine transporter (DAT) has been characterized as the primary mechanism by which cocaine produces its psychomotor stimulant actions. In order to understand further the molecular mechanisms underlying the pharmacological actions of cocaine, as well as mechanisms that underlie its abuse, structure-function studies have been directed toward characterizing the DAT protein at a molecular level. The design, synthesis and evaluation of 3-alpha-(diphenylmethoxy)tropane (benztropine) analogs have provided potent and selective probes for the DAT. Structure-activity relationships (SAR) have been developed that contrast with those described for cocaine, despite significant structural similarity. Furthermore, behavioral evaluation of many of the benztropine analogs, in animal models of cocaine abuse, has suggested that these two classes of tropane-based dopamine uptake inhibitors have distinct pharmacological profiles. In general, our previous studies have shown that the benztropine analogs, do not demonstrate efficacious locomotor stimulation in mice, do not fully substitute for a cocaine discriminative stimulus and are not appreciably self-administered in rhesus monkeys. These compounds are generally more potent than cocaine as dopamine uptake inhibitors, in vitro, although their actions in vivo are not consistent with this action. These observations suggest that differing binding profiles at the serotonin and norepinephrine transporters as well as at muscarinic receptors might have significant impact on the pharmacological actions of these compounds. In addition, by varying the structures of the parent compounds and thereby modifying their physical properties, pharmacokinetics as well as pharmacodynamics will be directly effected. Therefore, in an attempt to systematically evaluate the impact of chemical modification on these actions, a series of N-substituted (H, CH3, allyl, benzyl, propylphenyl and butylphenyl) analogs of 3'-chloro-, 4'-chloro- and 4,4"-dichloro-3-alpha-(diphenylmethoxy)tropanes were synthesized. These compounds were evaluated for binding to the dopamine, serotonin and norepinephrine transporters and to muscarinic m1 receptors. SAR were developed and compared to a series of previously reported N-substituted-benztropines. In another series of compounds, based on the sigma receptor antagonist, rimcazole, a novel series of [3-cis-3,5-dimethyl-1-piperazinyl)alkyl]bis-(4'-fluorophenyl)amines were designed and synthesized. We found that substitution of the carbazole ring system of rimcazole with bis-(4'-fluorophenyl)amine improves binding affinity and selectivity for the DAT. The most potent compound in this series showed a DAT binding affinity (Ki=17.6 nM) comparable to GBR 12909. Despite high affinity binding at DAT, and structural similarity to GBR 12909, preliminary studies suggest that this compound behaves more like rimcazole than GBR 12909 and does not demonstrate a cocaine-like behavioral profile in mice. Further synthetic and behavioral studies are underway to develop these agents, in both structural classes, as potential cocaine abuse therapeutics. In addition to developing agents for in vivo studies, we have also synthesized a number of important molecular tools in the form of radioactive and/or irreversible ligands. Currently, the most commonly utilized radioligand for the DAT is [3H]WIN 35,428. Although this ligand has been useful for binding studies involving animal brain tissue, it has a relatively low affinity of (Kd=40-50 nM) and is thus less than ideal for binding studies with adherent heterologous cells expressing DAT. We have recently developed [3H]MFZ 2-12, a novel, high affinity, tritiated ligand for the DAT. It has ~20-fold higher affinity than [3H]WIN 35. Thus at a ligand concentration of 2.5 nM, ~50% occupancy is achieved with [3H]MFZ 2-12 in contrast to ~6% by [3H]WIN35,428. Since the nonspecific binding of these two ligands is a similar fraction of the total added counts and since they are of similar specific activity, at 2.5 nM the signal and the signal to noise ratio achieved with [3H]MFZ 2-12 are approximately 10-times that observed with [3H]WIN 35,428. For these reasons, [3H]MFZ 2-12 is better suited for saturation analysis, filtration assays, and use with adherent cells expressing hDAT. In addition, to follow up on previous studies with the benztropine-based photoaffinity label, GA 2-34, which covalently attached to the DAT in a distinctive transmembrane domain region from a cocaine-based photolabel, RTI 82, two novel irreversible ligands were designed and synthesized. Both products bound to DAT with comparable potency (IC50=30 nM) to the cocaine analogue, RTI 82 and demonstrated wash resistant displacement of [3H]WIN 35,428 in HEK 293 cells stably transfected with hDAT. These ligands have recently been radioiodinated to be used in immunologic and peptide mapping studies to further characterize the binding domains for tropane-based dopamine uptake inhibitors at the DAT. In addition to classical drug design and synthesis, we have continued our computational chemistry efforts with a three dimensional quantitative structure-activity relationship (3D-QSAR) study on a series of mazindol analogues using the comparative molecular field analysis (CoMFA) method with their corresponding DAT binding affinities. The cross-validated CoMFA models were derived from a training set of 50 compounds, and the predictive ability of the resulting CoMFA models was evaluated against a test set of 21 compounds. A set of alignment rules was derived to superimpose these compounds onto a template structure, mazindol. These CoMFA models yielded significant cross-validated r2cv values. The best QSAR model was selected based on the predictive ability of the activity on the external test set of compounds. The analysis of coefficient contour maps provided further insight into the binding interactions of mazindol analogues with the DAT.